If the effects of an external stimulus such as ligand binding, environmental change in temperature or pH, mechanical or electrical stimulus, and the like on living cells can be observed in real time in an alive state, it would be possible to measure more accurately the effect of various active substances or physical conditions exerted on the cells, greatly contributing to a rapid diagnosis and determination of a therapeutic strategy in clinical sites. It is accordingly desired to develop an apparatus or diagnostic system for allowing the evaluation of the physiological activity on such cells since there is no device or system available for clinical tests until now.
For example, stimulation of mast cells results in aggregation of high-affinity IgE receptors at the first stage, followed by phosphorylation of α- and β-chains within several seconds. These changes induce association and phosphorylation of a variety of kinase proteins to activate a series of enzymes involved in the intracellular signal transduction such as tyrosine kinase, phospholipase C or G protein, causing a release of a chemical transmitter by infusion of secretory granules to cell membranes after several to several ten minutes. It is known that, simultaneously, a transcription factor is activated, and after a lapse of several to several ten hours, proinflammatory cytokines such as TNFα and IL-4 are synthesized and released.
So far, the dynamic behavior of cellular degranulation has been measured by recovering the supernatant of the reaction medium every time the analysis required to determine the content of the materials such as histamine and cytokines contained therein. In this method, however, it was necessary to recover at least a part of the supernatant of the reaction mixture for measurement and to suspend the reaction simultaneously with the measurement because the reaction condition be easy to change.
In addition, dynamics of the intracellular calcium concentration, pH change, intracellular transfer of a particular protein, and so on, can be analyzed by modification of the cells by loading of a specific fluorescent probe or by transfection with a protein containing a GFP tag. In these methods, however, some preliminary modification of the cells is required. Thus, a problem comes that there is a possibility that the cellular function will be influenced at least by the degree of the modification and in principle the cells used in these tests cannot be turned back to the organisms.
On the other hand, in an SPR (surface plasmon resonance) analyzer, it is possible to conduct measurement of the reaction and binding amount between biomolecules such as proteins as well as a kinetic analysis thereof by observing a change of a resonance angle in real time utilizing the surface plasmon resonance phenomenon. The change of a resonance angle depends on a change of permittivity in the vicinity of a gold film on a sensor. The one end of a protein to be measured for the binding is first immobilized on the gold film. Proteins each has an intrinsic permittivity. In SPR, the binding of a ligand to a protein fixed on the gold film is measured. When the binding of the ligand to the protein occurs, the complex is formed on the gold film and the permittivity will be changed. That is, information on the amount of the binding between biomolecules, kinetic constant of binding, kinetic constant of dissociation, dissociation constant, affinity constant, and the like, can be obtained by following up the permittivity of the surface of the gold film in real time.
An alternative device has been developed in which the amount of a ligand binding to the fixed living cells, kinetic constant of binding, kinetic constant of dissociation, dissociation constant, affinity constant, and the like can be measured in addition to the biomolecules such as proteins.
As mentioned above, an SPR analyzer in which a living cell can be targeted, permits measurement of the change of permittivity during exposure of the cell to an external stimulus. This time, the present inventors have found that the reaction of cells per se caused by an external stimulus makes the permittivity change.
The present application has been filed in view of the above-mentioned situation for the purpose of providing a new method for evaluating accurately the level of the physiological activity of an external stimulus on living cells.